2.1 MAb Expression
Monoclonal antibodies hold great promise for application in a wide range of diagnostic and therapeutic (clinical) settings, as evidenced by current clinical use of monoclonal antibody-derived products for transplantation, tumor imaging, therapeutics and diagnostics.
Currently two main methods used for commercial monoclonal antibody (“MAb”) production are generally employed; in vivo mouse ascites fluid and in vitro cultivation of hybridoma cell lines. The production of a MAb in vivo from mouse ascites fluid is limited in that it produces solid tumors in mice and results in death of the animal and low-level yields of MAbs. In vitro cultivation of hybridoma cell lines also has limitations. For example, it has been estimated that a minimum of 1000 clones need to be screened to find just two MAb-producing hybridoma cell lines. Most clones are not considered to be useful because of inappropriate specificity. In addition, after going through several passages, hybridoma cell lines may lose certain chromosomes and stop producing the MAb. Recombinant production of MAbs could avoid some of the problems associated with production of MAbs from hybridoma cell lines and ascites fluid.
A large number of heterologous single chain polypeptides have been produced by host cells transformed by recombinant DNA techniques. However, very few functional multichain polypeptides have been successfully produced by recombinant techniques. Recombinant dimeric polypeptides have been synthesized as a single chain polypeptide, coded for by a single DNA sequence, which is then cleaved in the host cell subsequent to synthesis to form the dimeric structure. In some cases the polypeptide chains are synthesized separately and then assembled after isolation from the host cell. Disadvantages of recombinant protein production in E. coli include inefficient secretion, formation of insoluble protein complexes in inclusion bodies, the presence of endotoxin, lack of glycosylation, and lack of N-terminal methionine processing (see Buchner, Anal. Biochem. 205:263 (1992)), which often affect the functionality of the recombinant protein, or hinder efficient and cost-effective production and purification.
A number of heterologous proteins have been expressed in yeast. Examples include interferon (U.S. Pat. No. 4,775,622, Hitzeman, et al., Nature, 292, 717, 1991); platelet derived growth factor (U.S. Pat. No. 4,801,542); and glyceraldehyde-3-phosphate dehydrogenase (Holland et al., Basic Life Science, 19:291, (1981)). Burke et al., U.S. Pat. No. 4,876,197 discloses a DNA construct comprising a transcription regulatory region obtained from the yeast ADH2, the regulatory region of acid phosphatase (PHO5) or GAL4 which provides for inducible transcriptional regulation, a transcriptional initiation region from the yeast glyceraldehyde-3-phosphate dehydrogenase gene (“TDH3”) and a terminator region.
The structure of antibody molecules and the nature of genes coding for them permit extensive manipulation and shuffling of antibody genes to produce recombinant antibodies with domains from different proteins and species. Such manipulation and shuffling can create MAbs with desired specificity, effector functions, reduced immunogenicity and/or binding sites for additional molecules. Recent advances in genetic engineering have made it possible to design and generate single chain, chimeric and humanized antibodies with desired specificities and binding sites (Vaughan et al., Nature Biotech. 16:535 (1998)).
Expression of recombinant MAbs using different expression systems such as bacteria, yeast, baculovirus and mammalian cells have been reported (Gen. Eng. News p. 12, August (1996)). Bacterial cells produce MAbs which accumulate as improperly folded, non-native proteins in inclusion bodies. However, the cell cultures are generally very low.
Humanized bispecific antibody produced from E. coli in secreted form was found to simultaneously bind different antigens on two different cells (Russoniello et al., Clin. Cancer Res. 4:2237 (1998)). Using Pichia pastoris, Ridder et al. have reported production of a soluble and functional rabbit single chain antibody fragment (“ScFv”) (Biotechnology 13:255-60, (1995)). The yields of ScFv for human leukemia inhibition factor was 100 mg/L. Glockhushuber et al. reported production of single chain and Fab fragments of antibodies in E. coli. (Biochem. 291362 (1990)). The yield in this system was poor (10-100 ug/ml) with the bacterial products being secreted in the periplasm and not glycosylated, requiring solubilization, denaturation, reduction, and renaturation to facilitate the formation of intramolecular disulfide bonds and the native conformation. Glockhushuber et al., Biochem. 29:1362 (1990). Another disadvantage of E. coli derived polypeptides is endotoxin contamination which can cause immune reactions in patients. In addition, E. coli do not have the ability to remove the N-formyl-methionine by post-translational modification which is required for the production of functional antibody formation. Glockhushuber et al., Biochem. 291362 (1990).
U.S. Pat. No. 4,816,397 (“the '397 patent”) describes the process for production of multichain polypeptides or proteins in a single host cell, which comprises transforming the host cell with DNA coding for each of the polypeptide chains. The invention also describes the production of recombinant IgG heavy and light chain or fragments thereof having an intact variable domain. While the '397 patent describes the production of both a heavy and light chain in a single cell, the expressed polypeptides were found in inclusion bodies in the bacterial cells in which they were produced and required cumbersome denaturation. Only a small fraction of the amount expressed was retrievable in functional, soluble form.
Feasibility of expression of functional immunoglobulin (IgG) in yeast was first reported by Wood et al. (Nature 314:446 (1985)) and Carlson (Mol. Cell Biol, 8:2638; 46, (1988)). Functional IgG against alcohol dehydrogenase was described using a yeast inducible promoter. Using GAL1-10 bidirectional promoter, Bowdish et al. (J. Biol Chem., 266:11901-8 (1991)) produced properly folded Fab fragment of a catalytic antibody, permitting the expression of low levels of two antibody polypeptides simultaneously. However, the expression of heavy chain gene was more efficient than that of light chain gene from GAL1 10. The results of Bowdish et al. indicate that recombinant heavy chain polypeptides are reasonably stable in yeast cytoplasm. Typically 100-200 ug/L of Fab was expressed which accounted for approximately 0.1% of total cellular protein. In comparison to the prior art methods, an advantage of the yeast expression system of the present invention is that it can simultaneously express two proteins (or protein subunits) in similar amounts, thereby favoring higher yields of functional multichain molecules.
In addition, recombinant MAbs have been expressed in hybridoma or myeloma cell lines. See David Robinson, Biotech Bioeng. 55:783 (1997). The current methodologies are limited by a low secretion rate of cell lines and the difficulties of selecting human clones secreting IgG. See Bobbington et al., Biotech 10:169 (1992). The media contain as much as 50 ingredients, and can take up to 14 days for fermentation making development of a mammalian cells secreting MAbs slow.
In some cases the polypeptides produced by the aformentioned techniques are not immunologically functional as they are incapable of combining with complementary heavy or light chains to provide functional IgG molecules.